Project Summary/Abstract Replication of HIV-1, the causative agent of Acquired Immune Deficiency Syndrome (AIDS), involves the assembly of immature particles composed of the Gag polyprotein and subsequent maturation of these particles by proteolytic cleavage. Although HIV-1 infection can be effectively controlled through the judicious administration of antiviral drugs, therapy is not curative, and drug resistance is a constant concern. Axiomatically, HIV-1 depends on interactions with host cell molecules at every stage of its replication cycle. Although many of these virus-host interactions occur between proteins, the host cell metabolite inositol hexakisphosphate (IP6) has recently emerged as a key host molecule involved in HIV-1 replication. IP6 appears to bind to the Gag polyprotein in infected cells, thus stabilizing the Gag hexameric lattice and promoting virion assembly. Remarkably, IP6 also binds to the cleaved viral CA protein in vitro and promotes CA self-assembly into cone- like structures that are morphologically similar to native HIV-1 capsids. Based on these observations, a model has been proposed in which IP6 is released upon cleavage of the Gag lattice by the viral protease during HIV-1 maturation. Release of IP6 permits its binding to assembling CA hexamers, thus stabilizing the mature capsid lattice. In this project, we will validate key predictions of this model. Employing novel and sensitive assays to quantify the levels of IP5 and IP6 associated with purified subviral complexes, we will identify the specific cleavages in the Gag polyprotein required for dissociation of these ligands from the immature Gag lattice. Second, we will identify the molecular determinants of IP6 binding to the mature capsid lattice, including testing the role of Arg18 in CA that has been shown to form ionic interactions with IP6 in vitro. Finally, we will quantify the levels of IP6 present in particles of diverse retroviruses as a first step in understanding the range of retroviruses that utilize IP6 in their replication cycles. IP6 is the first non-nucleotide host cell metabolite on which HIV-1 replication has been shown to depend. Defining the mechanism of IP6 action in HIV-1 maturation is essential to understand how HIV-1 exploits this novel virus-host interaction. Ultimately, the project may lead to the development of new antiviral drugs, thus expanding the available therapeutic options for the long-term management of HIV-1 infection.